Liquid chromatography is one of the most powerful and commonly-used techniques available today for the separation of materials from solutions. Examples of materials removed from solution using liquid chromatography include proteins, viruses, nucleic acids, pyrogens, fine chemicals, food additives, drugs and the like. As can readily be appreciated, for many such applications, it is highly undesirable for any portion of the chromatography column to become colonized by and/or contaminated with microbes since such microbes may affect the purity of any material prepared using the column.
In response to this concern, rather elaborate steps have been taken in the past in an attempt to establish and maintain sterile conditions within chromatography columns. Such steps have included autoclaving and/or chemically sterilizing chromatography resins ex situ (this option being limited to those resins which are amenable to such forms of treatment), pre-sterilizing chromatography tubes and their associated parts by chemical, thermal and/or radiative means, packing chromatography resins into columns in environments of low germ concentration, and pre-sterilizing sample solutions and aseptically connecting them to the columns.
Despite the array of sterilization procedures mentioned above, the maintenance of sterile conditions in chromatography columns is difficult to achieve in practice because chromatography columns typically include one or more "dead spaces" wherein microbes have a tendency to become ensconced and which cannot readily be accessed in situ by washing the column with a suitable sterilization solution.
For example, referring to FIG. 1, there is shown a fragmentary, simplified, schematic, section view of a first type of conventional liquid chromatography column, the first conventional chromatography column being represented generally by reference numeral 11. Column 11, which may be, for example, a Sepracor UPSCALE.TM. chromatography column (Marlborough, Mass.), includes a glass chromatography tube 13 which is partially filled with a suitable chromatography resin (not shown). A piston assembly 15 comprising an upper portion 15-1 and a lower portion 15-2 is mounted inside the upper portion of tube 13. The outer peripheral edge 17 of lower portion 15-2 extends conically downwards and, in combination with the inside surface of tube 13 and upper portion 15-1, defines an annular space 19. A sealing ring 21 is positioned within space 19 and extends downwardly just beyond the bottom of lower portion 15-2.
Lower portion 15-2 of piston 15 is also shaped to include radial grooves 22, a centrally-located fluid distribution channel 23, and a downwardly-extending collar 24. Collar 24 is shaped to define a circular orifice 25 in fluid communication with channel 23 and includes a pair of annularly-shaped, spaced-apart slots 29 and 31. An expanding ring 33 of resilient material is mounted in slot 29, and an O-ring 35 is mounted in slot 31. A polyethylene frit 37 (or gauze membrane), which is used both to distribute the liquid transmitted through channel 23 over the entirety of orifice 25 and to retain the resin in the column, is positioned across orifice 25 and is held in place between expanding ring 33 and O-ring 35.
As can be appreciated, numerous dead spaces not readily accessible to rapid liquid exchange using a sterilization solution are present in the areas where expanding ring 33 and O-ring 35 are joined to lower portion 15-2. Additional dead spaces are present in the areas between polyethylene frit 37 and expanding ring 33 and in the areas between polyethylene frit 37 and O-ring 35. Still further dead spaces are present within polyethylene frit 37, itself, which lacks well-defined pores for fluid flow.
Referring now to FIG. 2, there is shown a fragmentary, simplified, schematic, section view of a second type of conventional liquid chromatography column, the second conventional chromatography column being represented generally by reference numeral 51. Column 51 may be, for example, a BPG 100/500 BioProcess.TM. Glass Column chromatography column from Pharmacia (Uppsala, Sweden). Column 51 includes a glass chromatography tube 53 which is partially filled with a suitable chromatography resin (not shown). A piston assembly 55 comprising an upper portion 55-1 and a lower portion 55-2 is mounted inside the upper portion of tube 53. A sealing O-ring 57 is sandwiched between upper portion 55-1 and lower portion 55-2 of piston 55, O-ring 57 pressing against tube 53 and pressing upper and lower portions 55-1 and 55-2, respectively, of piston 55 together.
Lower portion 55-2 of piston 55 is shaped to include a centrally-located fluid distribution channel 63 which opens, at the base of lower portion 55-2, into a large orifice 65. A gauze membrane 67 is positioned over orifice 65 and is welded or glued onto a plastic ring 68 which, in turn, fits on a nut 69 mounted in lower portion 55-2. A rough net plastics disk 70 is positioned between piston 55-2 and membrane 67.
As can be seen, the fit of ring 68 on piston 55-2 creates a lot of dead spaces which cannot readily be accessed by washing the column with a sterilization solution. In addition, numerous dead spaces are present within membrane 67 and disk 70.